Summary Abstract Clostridium difficile causes diseases in humans ranging from antibiotic-associated diarrhea to potentially fatal pseudomembranous colitis. It is well known that C. difficile disease symptoms are largely mediated by the secreted cytotoxins produced during infection and that differentiation into metabolically dormant spores is critical to transmission between hosts. However, much of the basic biology of this organism remains poorly understood, including how C. difficile regulates adaptation to the host intestinal environment in response to extracellular stimuli. Our long-term goal is to characterize the nucleotide second messenger signaling pathways controlling virulence and transmission of C. difficile. The functions of the second messenger 3?,5? cyclic diadenylic acid (c- di-AMP) in C. difficile are completely unknown. In other bacterial species, c-di-AMP regulates growth, cell wall homeostasis, antibiotic resistance, osmotolerance, potassium transport, metabolism, sporulation and/or immune modulation. Preliminary data support a role for c-di-AMP in regulating C. difficile sporulation and other pathways potentially affecting disease. Our central hypothesis is that c-di-AMP has broad effects on C. difficile physiology through regulation of gene expression and protein function, with consequent effects on pathogenicity. We will test our hypothesis by (1) using transcriptional and proteomic approaches to define the c-di-AMP regulon and identify targets of c-di-AMP regulation, and (2) determining the impact of altering c-di-AMP on sporulation and germination in vitro and virulence and transmission in vivo. These studies will determine the capacity of c-di- AMP to influence the transmissibility and disease potential of C. difficile, and the findings will serve as the basis of future work characterizing the c-di-AMP signaling pathway in C. difficile. Ultimately, these studies may identify novel targets for inhibition of C. difficile virulence and transmission, which will help drive the much-needed development of additional treatment and prevention options.